The present invention is directed to peptide nucleic acids (PNAs) wherein naturally-occurring nucleobases or non-naturally-occurring nucleobases are covalently bound to a polyamide backbone. One preferred naturally-occurring nucleobase is 2,6-diaminopurine. Some PNAs of the present invention comprise at least one 2,6-diaminopurine nucleobase while other PNAs comprise at least one 2,6-diaminopurine nucleobase and at least one C1-C8 alkylamine side chain. The PNAs of the invention bind DNA with enhanced binding affinity and exhibit higher sequence specificity.
The function of a gene starts by transcription of its information to a messenger RNA (mRNA). By interacting with the ribosomal complex, mRNA directs synthesis of the protein. This protein synthesis process is known as translation. Translation requires the presence of various cofactors, building blocks, amino acids and transfer RNAs (tRNAs), all of which are present in normal cells.
Most conventional drugs exert their effect by interacting with and modulating one or more targeted endogenous proteins, e.g., enzymes. Typically, however, such drugs are not specific for targeted proteins but interact with other proteins as well. Thus, use of a relatively large dose of drug is necessary to effectively modulate the action of a particular protein. If the modulation of a protein activity could be achieved by interaction with or inactivation of mRNA, a dramatic reduction in the amount of drug necessary, and the side-effects of the drug, could be achieved. Further reductions in the amount of drug necessary and the side-effects could be obtained if such interaction is site-specific. Since a functioning gene continually produces mRNA, it would be even more advantageous if gene transcription could be arrested in its entirety. Oligonucleotides and their analogs have been developed and used as diagnostics, therapeutics and research reagents. One example of a modification to oligonucleotides is labeling with non-isotopic labels, e.g., fluorescein, biotin, digoxigenin, alkaline phosphatase, or other reporter molecules. Other modifications have been made to the ribose phosphate backbone to increase the resistance to nucleases. These modifications include use of linkages such as methyl phosphonates, phosphorothioates and phosphorodithioates, and 2xe2x80x2-O-methyl ribose sugar moieties. Other oligonucleotide modifications include those made to modulate uptake and cellular distribution. Phosphorothioate oligonucleotides are presently being used as antisense agents in human clinical trials for the treatment of various disease states. Although some improvements in diagnostic and therapeutic uses have been realized with these oligonucleotide modifications, there exists an ongoing demand for improved oligonucleotide analogs.
In the art, there are several known nucleic acid analogs having nucleobases bound to backbones other than the naturally-occurring ribonucleic acids or deoxyribonucleic acids. These nucleic acid analogs have the ability to bind to nucleic acids with complementary nucleobase sequences. Among these, the peptide nucleic acids (PNAs), as described, for example, in WO 92/20702, have been shown to be useful as therapeutic and diagnostic reagents. This may be due to their generally higher affinity for complementary nucleobase sequence than the corresponding wild-type nucleic acids.
PNAs are compounds that are analogous to oligonucleotides, but differ in composition. In PNAs, the deoxyribose backbone of oligonucleotide is replaced by a peptide backbone. Each subunit of the peptide backbone is attached to a naturally-occurring or non-naturally-occurring nucleobase. One such peptide backbone is constructed of repeating units of N-(2-aminoethyl)glycine linked through amide bonds.
PNAs bind to both DNA and RNA and form PNA/DNA or PNA/RNA duplexes. The resulting PNA/DNA or PNA/RNA duplexes are bound tighter than corresponding DNA/DNA or DNA/RNA duplexes as evidenced by their higher melting temperatures (Tm). This high thermal stability of PNA/DNA(RNA) duplexes has been attributed to the neutrality of the PNA backbone, which results elimination of charge repulsion that is present in DNA/DNA or RNA/RNA duplexes. Another advantage of PNA/DNA(RNA) duplexes is that Tm is practically independent of salt concentration. DNA/DNA duplexes, on the other hand, are highly dependent on the ionic strength.
Homopyrimidine PNAs have been shown to bind complementary DNA or RNA forming (PNA)2/DNA(RNA) triplexes of high thermal stability (Egholm et al., Science, 1991, 254, 1497; Egholm et al., J. Am. Chem. Soc., 1992, 114, 1895; Egholm et al., J. Am. Chem. Soc., 1992, 114, 9677).
In addition to increased affinity, PNAs have increased specificity for DNA binding. Thus, a PNA/DNA duplex mismatch show 8 to 20xc2x0 C. drop in the Tm relative to the DNA/DNA duplex. This decrease in Tm is not observed with the corresponding DNA/DNA duplex mismatch (Egholm et al., Nature 1993, 365, 566).
A further advantage of PNAs, compared to oligonucleotides, is that the polyamide backbone of PNAs is resistant to degradation by enzymes.
Considerable research is being directed to the application of oligonucleotides and oligonucleotide analogs that bind to complementary DNA and RNA strands for use as diagnostics, research reagents and potential therapeutics. For many applications, the oligonucleotides and oligonucleotide analogs must be transported across cell membranes or taken up by cells to express their activity.
PCT/EP/01219 describes novel PNAs which bind to complementary DNA and RNA more tightly than the corresponding DNA. It is desirable to append groups to these PNAs which will modulate their activity, modify their membrane permiability or increase their cellular uptake property. One method for increasing amount of cellular uptake property of PNAs is to attach a lipophilic group. U.S. application Ser. No. 117,363, filed Sep. 3, 1993, describes several alkylamino functionalities and their use in the attachment of such pendant groups to oligonucleosides.
U.S. application Ser. No. 07/943,516, filed Sep. 11, 1992, and its corresponding published PCT application WO 94/06815, describe other novel amine-containing compounds and their incorporation into oligonucleotides for, inter alia, the purposes of enhancing cellular uptake, increasing lipophilicity, causing greater cellular retention and increasing the distribution of the compound within the cell.
U.S. application Ser. No. 08/116,801, filed Sep. 3, 1993, describes nucleosides and oligonucleosides derivatized to include a thiolalkyl functionality, through which pendant groups are attached.
Peptide nucleic acids may contain purine or pyrimidine nucleobases. However, previous PNAs having a high purine nucleobase content exhibit decreased solubility at physiological pH. PNAs of the present invention overcome this problem.
Despite recent advances, there remains a need for a stable compound that enhances or modulates binding to nucleic acids, stabilizes the hybridized complexes and increases the aqueous solubility.
The present invention provides peptide nucleic acids (PNAs) with higher binding affinity to complementary DNA and RNA than corresponding DNA. The PNAs of the present invention comprise ligands linked to a polyamide backbone. Representative ligands include the four major naturally-occurring DNA nucleobases (i.e. thymine, cytosine, adenine and guanine), other naturally-occurring nucleobases (e.g. inosine, uracil, 5-methylcytosine, thiouracil or 2,6-diaminopurine) or artificial bases (e.g. bromothymine, azaadenines or azaguanines) attached to a polyamide backbone through a suitable linker.
The present invention provides a peptide nucleic acid having formula (I): 
wherein:
each L is independently selected from a group consisting of naturally-occurring nucleobases and non-naturally-occurring nucleobases, at least one of said L being a 2,6-diaminopurine nucleobase;
each R7xe2x80x2 is independently hydrogen or C1-C8 alkylamine;
Rh is OH, NH2 or NHLysNH2;
Ri is H, COCH3 or t-butoxycarbonyl; and
n is an integer from 1 to 30.
Preferably, R7xe2x80x2 is C1-C8 alkylamine. More preferably, R7xe2x80x2 is C3-C6 alkylamine. Even more preferably, R7xe2x80x2 is C4-C5 alkylamine. Still more preferably, R7xe2x80x2 is butylamine.
Preferably, at least one of the R7xe2x80x2 is C3-C6 alkylamine. More preferably, at least one of the R7xe2x80x2 is C4-C5 alkylamine. Still more preferably, at least one of the 7xe2x80x2R is butylamine. Even more preferably, substantially all of the R7xe2x80x2 is butylamine.
Preferably, the carbon atom to which substituent R7xe2x80x2 are attached is stereochemically enriched. Hereinafter, xe2x80x9cstereochemically enrichedxe2x80x9d means that one stereoisomer is present more than the other stereoisomer in a sufficient amount as to provide a beneficial effect. Preferably, one stereoisomer is present by more than 50%. More preferably, one stereoisomer is present by more than 80%. Even more preferably, one steroisomer is present by more than 90%. Still more preferably, one stereoisomer is present by more than 95%. Even more preferably, one stereoisomer is present by more than 99%. Still even more preferably, one stereoisomer is present in substantially quantitatively. Preferably, the stereochemical enrichment is of R configuration.
Preferably, the peptide nucleic acid of the present invention is derived from an amino acid. More preferably, the peptide nucleic acid of the present invention is derived from D-lysine.
The PNAs of the present invention are synthesized by adaptation of standard peptide synthesis procedures, either in solution or on a solid phase.
The monomer subunits of the invention or their activated derivatives, protected by standard protecting groups, are specially designed amino acids.
The present invention also provides a compound having formula (II): 
wherein:
L is a 2,6-diaminopurine nucleobase;
R7xe2x80x2 is hydrogen or C1-C8 alkylamine;
E is COOH or an activated or protected derivative thereof, and
Z is NH2 or NHPg, where Pg is an amino-protecting group.
Preferably, R7xe2x80x2 is C3-C6 alkylamine. More preferably, R7xe2x80x2 is C4-C5 alkylamine. Still more preferably, R7xe2x80x2 is butyl amine.
Preferably, the carbon atom to which substituent R7xe2x80x2 is attached (identified by an asterisks) is stereochemically enriched. Preferably, the stereochemical enrichment is of R configuration.
Preferably, compound (II) of the present invention is derived from an amino acid. More preferably, compound (II) of the present invention is derived from D-lysine.
The present invention also provides a pharmaceutical composition comprising peptide nucleic acids of the present invention and at least one pharmaceutically effective carrier, binder, thickener, diluent, buffer, preservative, or surface active agent.
The present invention further provides methods for enhancing the solubility, sequence specificity and binding affinity of peptide nucleic acids by incorporating 2,6-diaminopurine nucleobases in the PNA backbone.